Use of liver growth factor (lgf) as a neural tissue regenerator

ABSTRACT

The invention comprises the use of LGF in the production of medicinal products that can be used in the pleiotropic tissue regeneration of one or more damaged tissues, in which at least one of the damaged tissues forms part of the central nervous system and the medicinal product is intended to be administered systemically. The invention is based on the fact that intraperitoneal administration of LGF can promote a positive effect on a Parkinson&#39;s disease model. As such, in a preferred embodiment of the invention, the medicinal product is intended for the treatment of Parkinson&#39;s disease, particularly when the medicinal product is intended for humans.

FIELD OF TECHNOLOGY

The invention relates to the contribution of the use of liver growthfactor (LGF) for the manufacture of a medicinal product for systemicadministration in mammals for the treatment of a neurodegenerativedisease, treatment of which requires tissue regeneration. Said inventionrepresents an improvement of the invention presented in patentapplication ES200601018 and in international patent applicationPCT/ES2007/070080, in which the priority of the foregoing was claimed.

PRIOR ART

Liver growth factor (LGF), which was discovered some years ago (Díaz Gilet al., 1986), is a molecule purified from rat serum after 70% partialhepatectomy, or in rats with ligation of the biliary duct which, wheninjected in rats or mice, has activity “in vivo” as a growth factor,increasing the synthesis of liver DNA, the dry weight of the liver, thenumber of “PCNA-positive” cells (PCNA: proliferating cell nuclearantigen), producing transient hyperplasia with neither immediate norpermanent aggressive effects being detected: without production offibrosis, amyloids, or mitochondrial or nuclear disorders (Díaz Gil etal., 1994).

Its chemical structure was defined by the same authors as analbumin-bilirubin complex, after investigation of absorption spectra,fluorescence, circular dichroism, tryptic maps, amino acid composition,electrophoretic mobility, immunofluorescence, formation ofalbumin-bilirubin complexes “in vitro”, investigation of biologicalactivity both “in vivo” and “in vitro” and identification by HPLC (DíazGil et al., 1987; Díaz Gil et al., 1988).

LGF also has activity “in vitro”, in a primary culture of rathepatocytes, increasing the synthesis of DNA, the cell count, theactivity of the membrane transport system A and others (Díaz Gil et al.,1986). LGF has also been purified from serum of humans with type Bhepatitis, with structure and characteristics almost identical to thatfrom rats (Díaz Gil et al., 1989). Other authors (Abakumova et al.,1994) have confirmed the activity of albumin-bilirubin complexes asliver growth factors.

Furthermore, it has been demonstrated that LGF is able to stimulate theregeneration of the liver damaged by the action of various hepatotoxins(Díaz Gil et al., 1994b; Díaz Gil et al., 1999). In a model of cirrhosisinduced by CCI₄, once an irreversible situation had been reached,injection of LGF was able to reduce fibrosis, producing a substantialremodelling of the hepatic parenchyma, improvement of inflammation andnecrosis, increase in hepatic function and restoration of varioushaemodynamic functions, such as: portal pressure, arterial pressure,portosystemic shunting and systemic vascular resistance, as well asreduction of ascites. However, the extremely complex network that givesrise to the establishment of fibrosis in the various types of organs,although having various traits in common, has particular featuresdepending on the organ in question, such that the antifibrotic action ofLGF in the liver was not guaranteed to display the same ability in someother different organ.

In addition, and in research conducted by various groups principally onendothelial cells, three types of albumin receptors have been identifiedon the cell membrane, the three glycoproteins: gp60, with the mainfunction of transcytosis, which principally passes albumin from one sideto the other in endothelial cells (Ghinea et al., 1988), and two withthe function of endocytosis, for taking up albumin into the cell, gp18and gp31. These last-mentioned receptors bind preferentially to albuminsin which their conformation has been modified for “binding” to a ligand.The affinity is 1000 times compared to what is observed with nativealbumin (Schnitzer et al., 1992). gp18 and gp31 are expressedpreferentially in endothelial cells of fetal tissues, neonates oradults, higher concentrations being recorded in organs with very activeproliferation or in phases of increased growth, in brain, lung, thymus,heart, skeletal muscle, liver, spinal cord, spleen, pancreas, testes,adenohypophysis, placenta, endometrium, myometrium and leukocytes(Morioanu et al., 1990).

The hypothesis used by these authors to explain the reasons for theuniversality and abundance of these receptors gp18 and gp31 in organs ofalmost every type is that they either serve: 1) to metabolize “modifiedalbumins” (albumin with some other compounds bound to it, such as maleicanhydride, formaldehyde etc., which alters its conformation), which areknown to exist in human serum at varying concentrations, or 2) totransport nutrients necessary for growth of cells of almost every type.

Taking into account the abundance of the receptors gp18 and gp31 invarious tissues, their high concentration in organs with very activeproliferation and the fact that the natural ligands of gp18 and gp31 are“modified albumins” (albumins with a conformational change, the same ashappens with LGF, which is an albumin with a specific conformationalchange, connected with appearance of biological activity (see Díaz Gilet al., 1987)), the inventors thought that perhaps LGF might be able toact as a growth and regeneration factor in a great variety of tissues,just as they had demonstrated previously in the liver; finally, theydecided to test the hypothesis of the validity of LGF as a tissueregeneration factor of the pleiotropic type, which was novel withrespect to other strategies for use of LGF proposed so far. For example,the patent “Method for diagnosis and monitoring of hepatopathies bydetermination of liver growth factor in blood plasma and/or serum”,publication number 2005259, was granted in 1989, but LGF was notcontemplated as a possible therapeutic agent in the correspondingapplication.

The inventors demonstrated recently that the mitogenic action of LGF onthe liver is mediated, at least partly, by TNF-α (Díaz Gil et al.,2003). They took this as an indication that LGF could also havemitogenic action on other tissues in which said cytokine can beexpressed. However, the increase in TNF-a does not in itself producemitogenic effects; in fact, it is closely linked to the acute phase,regardless of its aetiology: ischaemia, trauma, inflammation, toxicity(as reviewed by Ding W X et al., 2004, and Trauner M et al., 1999) andeven when injected in rats it can give rise to endocrine andhaematologic disorders (Kettelhut I et al., 1987); moreover, it isclosely connected with cell death, both by necrosis and by apoptosis(reviewed by Malhi H et al., 2006). Since stimulation of TNF-α isassociated both with effects of mitogenic stimulation and with effectsof cell death, an increase in its expression induced by LGF does notdescribe the activity of LGF unambiguously. Moreover, LGF is able toinduce mitosis in a culture of hepatocytes (Díaz Gil et al., 1986), inwhich there are no endothelial cells that could produce TNF-α.Accordingly, its possible use for inducing proliferation of tissuesother than hepatic tissue and the effect that it might have on themremained to be demonstrated.

Following the line of the possible validity of LGF as a tissueregeneration factor, the inventors recently demonstrated the capacity ofLGF for reducing hypertension in hypertensive rats by producing adecrease in fibrosis in the carotid artery and increasing the cell countof the vascular smooth musculature but without altering the insidediameter or the average thickness of said arteries (Somoza et al.,2006).

They also demonstrated the capacity of LGF for increasing the number ofdoparninergic terminals in animal models of Parkinson's disease (ReimersD. et al., 2006). It was observed that LGF produces a notable increasein doparninergic terminals, as well as an improvement in behaviour(rotational test). These results opened up a promising route regardingthe possibility of using LGF in other fields of considerable interest.However, the experiments presented in this publication were conducted byadministering LGF to mice by the intrastriatal route, i.e. administeringit directly into the brain, without the need for it to cross theblood-brain barrier. Unfortunately this route of administration is notreally feasible in human beings, so it is difficult to use it in actualclinical practice for the treatment of Parkinson's disease in humanbeings.

The capacity of LGF, following intraventricular application, forpromoting the generation of new neurons and migration thereof into thedenervated striatum was also demonstrated recently (Gonzalo-Gobernado etal., 2007).

This same line of research was reinforced by the findings described inpatent application ES200601018. This describes the use of liver growthfactor in the manufacture of a medicinal product used for pleiotropictissue regeneration, based on the mitogenic effect in general and theangiogenesis stimulating effect in particular that LGF exerts on variousorgans, such as the testes, the spinal cord or the skin, as well as itsantifibrotic activity in coronary arteries, its capacity for remodellingthe walls of coronary arteries and arteries of the greater and lessercirculation, its ability to stimulate anticholesterolaemic enzymes suchas paraoxonase-1 and the inhibitory effect on the growth of livercarcinoma cells. These effects of LGF mean it can be used for thetreatment of pathologies such as atherosclerosis, coronary disease,thrombosis or liver carcinoma, as well as helping in the recovery ofdamaged tissues such as those of the testes, spinal cord, infarctedheart, fractured bone, damaged skin or any other tissue whose recoveryis accelerated by stimulation of angiogenesis.

The method of administration of LGF in the experiments relating toregeneration of the spinal cord was intraperitoneal injection of LGF.Two different situations were included: in the first of these, traumaticlesion was produced and fetal cells were transplanted two months later,LGF being injected immediately, 4 injections in 2 weeks. In the secondsituation, trauma or hemisection of the spinal cord was produced, LGFbeing injected one week later (4 injections in 2 weeks). Now, owing tothe characteristics of the models used, the blood-brain barrier (BSCB,blood spinal cord barrier) was profoundly altered as a consequence ofthe aggressive action to which it was submitted. This circumstance hasbeen extensively investigated by various authors: thus, the immediatedamage produced in the BSCB after mechanical trauma is known (Maikos andShreiber, 2007). To quantify this permeability, researchers have usedcompounds of low molecular weight, ¹⁴C-α-aminoisobutyric, increasedpermeability being detected 28 days after the trauma (Popovich et al.,1996), and compounds of higher molecular weight: for example by infusionof ¹⁴C-albumin (Wood et al., 1999; Pettersson et al., 1990; Sharma,2004), by injection of horseradish peroxidase, HRP (Jaeger et al.,1997), using luciferase (Whetstone et al., 2003), or techniques ofmagnetic resonance, MRI (Bilgen et al., 2001; Bilgen et al., 2002). Inaddition, loss of albumin at the site of the lesion was detected byimmunohistochemical methods (Gordh et al., 2006), even ten weeks afterthe trauma, a longer period than was used in the experiments in patentapplications ES200801121 and PCT/ES2007/070080. It can be concluded fromthe foregoing that the increase in permeability of the BSCB as a resultof trauma has been demonstrated by many authors, and is known by aperson skilled in the art, and occurs in both directions, from themedulla to the outside and vice versa, and that it is maintained for alonger time than that including injection of LGF in the second situationdescribed above. Additionally, in the first situation, LGF was injectedtwo months after the trauma, before transplant of fetal cells, butprecisely this fact also alters the permeability of the BSCB (Horner etal., 1996), this increased permeability having been detected up to twoweeks after the transplant.

Based on the foregoing, a person skilled in the art would consider thatthe positive effects of LGF observed in spinal cord regeneration wouldbe attributable to the increase in permeability of the blood-brainbarrier that is produced because the latter is altered, which makes itpossible for LGF to bind to the medulla without it being necessary thatthe intact blood-brain barrier is permeable to it.

Moreover, growth factors, in general, are not compounds that can easilycross the blood-brain barrier. Except in the case of IGF (insulin-likegrowth factor) (Reinhardt and Bondy, 1994) it is generally consideredthat the other growth factors do not cross this barrier. Although theability to cross the blood-brain barrier does not depend exclusively onsize, since LGF has a considerable molecular weight, 64 000 kDa, thismeans a priori that it is unlikely that it crosses the blood-brainbarrier.

Thus, the search for therapeutic agents that can be used forregeneration of damaged tissues belonging to the central nervous systemand accordingly the prevention and/or treatment of disorders or diseasesin which said tissues are involved, such as Parkinson's disease, is madedifficult, in general, by the problem of finding a compound that has therequired therapeutic activity and, at the same time, is able to crossthe blood-brain barrier, thus making possible its administration byroutes that can be applied in human beings, such as those providingsystemic administration (intravenous, intraperitoneal, intramuscularetc.).

The present patent application offers a solution to this problem.

BRIEF DESCRIPTION OF THE INVENTION

The present invention offers a solution to the problem of themanufacture of medicinal products designed to be administeredsystemically, directed at the treatment of neurodegenerative diseasesthat require the regeneration of damaged nerve tissue, it beingdemonstrated that LGF, administered systemically, is able to produceeffects in tissues of the central nervous system, which are, therefore,on the other side of the blood-brain barrier. Thus, the invention of thepresent application offers an improvement of the invention disclosed inapplication ES200601018, demonstrating the specific usability of LGF forthe regeneration of nerve tissue belonging to the central nervous systemeven when LGF is administered by a route that means that the desiredeffect must be produced on the other side of the blood-brain barrier.

The fact that LGF, administered systemically, is able to exert an effecton the other side of the blood-brain barrier, permits the treatment ofneurodegenerative diseases that afflict mammals in which the blood-brainbarrier is intact or minimally altered, without the need to supply themedicinal product by a route that requires direct administration to thecentral nervous system, such as the intracranial route: intrastriatal,intrathecal or intraventricular.

Thus, one object of the present invention is the use of liver growthfactor (LGF) in the manufacture of medicinal products for use in tissueregeneration of one or more damaged tissues, characterized in that atleast one of the tissues forms part of the central nervous system (CNS)and the medicinal product is designed to be administered systemically.

In a preferred embodiment of the invention, the medicinal product isdesigned for the treatment of a neurodegenerative disease. It isparticularly preferable for the medicinal product to be designed for thetreatment of Parkinson's disease, although other embodiments of theinvention are also possible in which the disease is selected from otherneurodegenerative diseases, such as Huntington's disease, ataxia andamyotrophic lateral sclerosis. Whatever the disease, it is preferredvery especially that the individual for which the medicinal product isdesigned is a human being.

In another preferred embodiment of the invention, the medicinal productis designed to be administered by the intravenous or intraperitonealroute.

The invention also relates to a method of treatment of a disorder ordisease whose severity or risk of deterioration decrease with theregeneration of one or more tissues, in which said disorder or diseaseis selected from those associated with the central nervous system, saidmethod comprising the administration of liver growth factor by thesystemic route to a human being or an animal. In a preferred embodimentof the method of the invention, LGF is administered by theintraperitoneal route. In another preferred embodiment of the method ofthe invention, LGF is administered by the intravenous route.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D show the effects of intraperitoneal injection of LGF in ratswith striatal lesion with 6-OHDA.

A shows, in various coronal sections, the percentage of the total areaof the striatum with TH-positive innervation in control animals (whitebar) and in animals treated with LGF (black bar). The distance, inmillimetres, from bregma to each of the seven levels of the striatum inwhich the coronal sections were made, is shown on the right of thediagram.

B shows the effect of saline (control) and LGF i.p. on the number ofdopaminergic neurons of the healthy (white bars) and damaged (blackbars) substantia nigra.

C shows the effect of the administration i.p of saline (control) or LGFon the incorporation of BrdU in the healthy (white bars) and damaged(black bars) striatum.

D shows the rotational behaviour, evaluated by the apomorphine test, ofthe control animals (empty circles) and those injected i.p. with LGF(black squares).

The results represent the mean value±SEM of 5 (B and C), 10 (A) or 15(D) independent animals. In A and B, *p≦0.05, **p≦0.01, ***p≦0.001 vscontrol animals. In B ***p≦0.001 vs healthy control side, ••p≦0.01 vshealthy LGF side. In C *≦p0.05, ***≦p0.01 vs damaged control side.

DETAILED DESCRIPTION OF THE INVENTION

As already mentioned, the closest precedent of the action of LGF on thebrain is a publication describing the action of LGF, administered byintrastriatal infusion, on rats with Parkinson's disease (Reimer et al.,2006). The present invention relates to the data of LGF in the samemodel of Parkinson's, this time administering the LGF by theintraperitoneal route (LGF has the same biological activity by theintravenous route). The difference in the route of administration of LGFand its activity in the brain is a key point in the invention describedin the present patent application and is closely associated with theconcept of blood-brain barrier (hereinafter blood-brain barrier).

The blood-brain barrier was discovered in 1885 by Paul Ehrlich, who byintravenous injection of the dye trypan blue in rabbits, observed thatall the organs were stained equally, apart from the brain. Eighty yearslater, it was discovered that the blood-brain barrier is constituted ofthe endothelial cells of the capillaries of the brain, which have verywell sealed connections and constitute a very effective physical barrier(Reese et al., 1967). Penetration through the blood-brain barrier is nota question of molecular size alone, since 98% of small molecules, withmolecular weight below 400, do not cross the blood-brain barrier.Furthermore, larger biotechnology products, such as monoclonalantibodies, recombinant proteins, compounds of “antisense” RNA or geneproducts do not cross the blood-brain barrier (see Pardridge, 2005 for areview). Similarly, a priori it seems unlikely that LGF, which is alarge molecule, can cross the blood-brain barrier.

Despite the large number of patients with CNS disorders and the smallnumber of molecules that cross the blood-brain barrier, fewer than 1% ofpharmaceutical companies have specific programmes for researchingproducts for solving this problem. The few components of low molecularweight for use in the brain have mainly focused on the treatment ofdepression, schizophrenia, chronic pain and epilepsy, and in the case ofParkinson's disease, only the administration of L-DOPA, a precursor ofdopamine (Lloyd et al., 1970) has been used. In an attempt to solve thisproblem, several strategies have been devised based on transportmediated by receptors, monoclonal antibodies used as “Trojan horses”,masking molecules with therapeutic action, such as recombinant proteins,antibodies, interference RNA and some others (see Pardridge, 2007, for arecent review).

The known activity of other growth factors that have demonstratedactivity when infused intracranially in experimental models ofParkinson's is closely related with this point: transforming growthfactor of the alpha type, TGF-α (Fallon et al., 2000), vascularendothelial growth factor, VEGF (Sun et al., 2003), glial cellline-derived neurotrophic factor, GDNF, or the combination of this andbrain-derived neurotrophic factor, BDNF (Torp et al., 2006). In allcases, although cerebral activity has been demonstrated by intracranialinfusion, it has not been demonstrated that they act by the intravenousor intraperitoneal route, possibly because they do not cross theblood-brain barrier.

Based on the foregoing, it would be expected that the action of LGF onthe brain by the intracranial route will not ensure that said LGF has abiological effect by the intravenous or intraperitoneal route when theblood-brain barrier is intact.

However, research subsequent to submission of patent applicationES200601018 demonstrated a surprising effect of the action of LGF,administered intraperitoneally, in the treatment of Parkinson's diseasein mammals with an intact blood-brain barrier, as is presented later inthe examples of the present specification, which give the results ofinjection of LGF by the intraperitoneal route.

Since the experiments relating to the administration of LGF forregeneration of the spinal cord, described in patent applicationES200601018, show that LGF is capable of regenerating the tissue of thedamaged spinal cord, it can be considered that LGF has the ability toregenerate, in general, nerve tissue of the central nervous system.Therefore its use can be extended to the manufacture of medicinalproducts for the treatment of any neurodegenerative disease thatrequires the regeneration of nerve tissue, in which the medicinalproduct is designed to be administered systemically, giving rise to animprovement of the pathologic situations described and a stimulatingeffect on regenerative processes in various tissues.

In addition, it is shown in Example 2 of the present patent applicationthat LGF, injected by the intraperitoneal route, is able to stimulatethe serotonin pathway in the brain in intact rats. Serotonin is aneurotransmitter with very varied functions, with decisive action andinfluence in many situations. Thus, it is known to participate in braindevelopment (Azmitia et al., 2007), in memory (Buhot et al., 2000), inAlzheimer's disease (Meltzer et al., 1998), in epilepsy (Bagdy et al.,2007), in sleep (Gao et al., 2002), in dementia (Yan et al., 2001), indepression (Grahame-Smith et al., 1992), in anorexia and bulimia (Kayeet al., 2005), and in many others (see Smythies et al., 2005 for areview).

Activation of the serotonin pathway promoted by LGF administered by theintraperitoneal route lends more support to the usefulness of LGF forneurodegenerative diseases other than Parkinson's disease, such asHuntington's disease, amyotrophic lateral sclerosis, ataxias, etc.

The fact that LGF is able to exert an effect on the central nervoussystem when administered systemically implies that, regardless of thespecific route of administration employed, whenever it has the resultthat LGF reaches the blood system and can reach the blood-brain barrierby the systemic route, the medicinal product will be able to produce atherapeutic effect on the desired neurodegenerative disease. Thereforethere are possible embodiments of the invention in which the medicinalproduct is designed to be administered by the intraperitoneal route, asin the examples of the invention, but also those in which the medicinalproduct is designed to be administered by the intravenous route.

Regarding the composition of the medicinal product, those are preferredthat include a pharmaceutically acceptable aqueous vehicle, in which theLGF is dissolved. Particularly saline medium, and very particularlyhypotonic saline medium, are preferred. In addition, the medicinalproduct can contain any pharmaceutically suitable excipient, such as,for example, one or more stabilizers. Another especially preferredpossibility is that in which the medicinal product includes LGF inlyophilized form, in a vacuum-sealed container, with optional additionalpresence of the saline vehicle in the same medicinal product; thispresentation makes it possible to prepare the LGF solution shortlybefore it is administered.

The present invention will now be explained in more detail on the basisof the Examples and Drawings that are presented below, which arecertainly not intended to limit the scope of the invention.

EXAMPLES Example 1 Effects of Intraperitoneal Administration of LGF onInduction of Axonal Growth and Neurogenesis

Since the results given in the article of Reimers et al. provide a basisfor the possible use of LGF in the treatment of Parkinson's disease, thepresent experiment was conducted to test whether, on administering LGFby a route that will mean that the effects tested must be evaluated onthe other side of the blood-brain barrier, positive effects were alsoobserved in a model of said disease.

Thus, to verify the possibility that LGF can be used as a therapeutictool administered by a route whose use is feasible in human beings, theeffects of intraperitoneal administration of LGF on the induction ofaxonal growth and neurogenesis were investigated in the experimentalmodel of Parkinson's disease described by Kirk et al. (1998).

The experimental model of Parkinson's disease used comprises theunilateral application of 4 injections of 6-hydroxydopamine (6-OHDA) atvarious points of the right striatum of female Sprague-Dawley ratsweighing 200-220 g. For this, a 10 μl Hamilton microsyringe was used, atan injection rate of 1 μl/min. After application of the neurotoxin, thecannula was left in place for a further 2 min before being withdrawnslowly. The dose and coordinates used were selected on the basis ofother works (Kirk et al., 1998). Concretely, the 6-OHDA was administeredat a concentration of 3.5 μg/μl, 2 μl per injection (7 μg of 6-OHDA perinjection). The stereotaxic coordinates (AP: anteroposterior; L:lateral; V: ventral) relative to the bregma (point on the surface of theskull at the junction of the coronal and digital sutures) and the dura,of each injection, were as follows:

-   -   1: AP: +1.3 mm; L: +2.6 mm; V: −5.0 mm    -   2: AP: +0.4 mm; L: +3.0 mm; V: −5.0 mm    -   3: AP: −0.4 mm; L: +4.2 mm; V: −5.0 mm    -   4: AP: −1.3 mm; L: +4.5 mm; V: −5.0 mm    -   In all cases, the incision bar was adjusted to 0 mm.

To determine the degree of lesion, a rotational behaviour test wasperformed at 15 days (subcutaneous injection of apomorphine 0.05 mg/kg).Injection of apomorphine induces rotational behaviour contralateral tothe lesion. Animals that rotated 100 or more times in 15 minutes wereconsidered to have lesions, and those that did not meet this criterionwere discarded. During the period prior to treatment with LGF, regularweekly rotation tests were conducted, in order to ensure completeestablishment of the lesion.

Eight weeks after the lesion, the animals were divided into two groups:treated with LGF (n=18) and treated with vehicle (n=15). Treatment withLGF was carried out as follows: LGF (5.0 μg/rat) was injectedintraperitoneally 6 times at intervals of 3 days (days 0, 3, 6, 9, 12and 15), maintained for an additional four weeks (the controls wereinjected with saline during this same time). At the time indicated, theanimals were sacrificed and were fixed by intracardiac perfusion withparaformaldehyde at 4% in PBS. The brains were frozen and 20-30 μmsections were prepared in the cryostat. The sections were incubated withantibodies specific for tyrosine hydroxylase (TH, ChemiconInternational, Temecula, Calif.), neurons (b-tubulin III, Bab Co,Richmond, Calif., and doublecortin, Chemicon International Inc.), glia(GFAP, DakoCytomation Inc., and O1, Sigma Chemical Co, St Louis, Mo.)and neural precursors (nestin, Development Studies Hybridoma Bank,University of Iowa, Ames, Iowa).

The results represent the mean value±SEM of 4 to 10 independent animals.Immunohistochemical analysis was performed using an epifluorescencemicroscope coupled to the CAST-GRID stereologic analysis software. Ineach animal, the fluorescence included in the area of the striatum ofhistologic sections previously selected by defined anteroposteriorcoordinates was evaluated. Statistical analysis was performed by one-wayANOVA followed by an unpaired t-test or the Bonferroni test for multiplecomparisons, and the differences were considered significant for p≦0.05.

1.1.—Effects of LGF on Dopaminergic Innervation

To determine the degree of degeneration of the nigrostriataldopaminergic terminals caused by lesion with 6-OHDA, a group of animals(n=4) was sacrificed at 8 weeks post-lesion. Immunohistochemicalanalysis of coronal sections from 7 different levels of the striatumshowed that approximately 30-40% of the surface of the striatumipsilateral to the lesion is innervated with fibres that areimmunopositive for the limiting enzyme in the synthesis ofcatecholamines, TH. Similar results were obtained in the group ofanimals sacrificed at 10 weeks post-lesion, which received 6intraperitoneal (i.p.) injections of vehicle. The intraperitonealadministration of LGF significantly increased TH-positive innervation atalmost all the levels of the striatum analysed, this effect being moremarked in the more anterior levels (FIG. 1A).

Parkinson's disease is characterized by loss of the dopaminergic neuronsof the substantia nigra (SN) that project into the striatum.Preservation of the neuronal damage exerted by 6-OHDA on these neuronsmight contribute to the increase in innervation observed in the animalstreated with LGF. However, the loss of TH-positive neurons in the SN ofthe animals treated with LGF was similar to that observed in the animalstreated with vehicle (FIG. 1B).

As already mentioned, LGF promotes neurogenesis in hemiparkinsonian rats(Gonzalo-Gobernado et al., 2007). To determine whether the generation ofnew dopaminergic neurons could contribute to the increase in theTH-positive innervation observed in the animals treatedintraperitoneally with LGF, a group of hemiparkinsonian rats received adaily intraperitoneal injection of 50 mg/kg of bromodeoxyuridine (BrdU)for three weeks, starting the treatment 24 hours after the firstinjection of vehicle or LGF. Intrastriatal lesion with 6-OHDA increasedthe incorporation of BrdU in the denervated striatum of these animals by300%. Immunohistochemical analysis of coronal sections from 1 level ofthe striatum (AP: −1.8) from the animals treated with vehicle showedsimilar results (FIG. 1C). However, intraperitoneal administration ofLGF did not affect proliferation, since the BrdU-positive cell count wassimilar in the healthy striatum and the denervated striatum of theseanimals (FIG. 1C). Independently of the changes in proliferationdescribed, comarkings in the striatal parenchyma of the BrdU-positivecells with neuronal markers (β tubulin III or doublecortin) and/or THwere not observed in any of the experimental groups investigated.Conversely, and although it was recently demonstrated that neurogenesisis stimulated in the SN of healthy rats and rats with lesion with 6-OHDA(Zhao et al., 2003), BrdU-/TH-positive cells were not observed in the SNof the animals treated with LGF.

It can be concluded from all these results that intraperitonealadministration of LGF, just like intrastriatal administration of thefactor, promotes the reappearance of the TH-positive terminals in thestriatum of rats with intrastriatal lesion with 6-OHDA.

1.2.—Effects of Intraperitoneal Administration of LGF on RotationalBehaviour

The previous works of the group of inventors indicate that LGFadministered in the denervated striatum of hemiparkinsonian ratspromotes a moderate improvement in the rotational behaviour induced byapomorphine (Bazán et al., 2005, Reimers et al., 2006). To determinewhether intraperitoneal administration of LGF is also able to restoremotor function, the rotations induced by apomorphine weekly before,during, and two weeks after the period of treatment with the factor,were evaluated in the rats with unilateral lesion of the striatum with6-OHDA.

As was demonstrated in FIG. 1D, the animals treated with vehicle did notshow significant changes in rotational behaviour induced by apomorphineduring the period of study. However, 4 weeks after the start oftreatment with LGF, a significant decrease in the number ofcontralateral turns induced by apomorphine in this experimental groupwas observed, and this effect was maintained during the four weekssubsequent to completion of the treatment with the factor.

We can therefore conclude that intraperitoneal administration of LGFstimulates the regeneration of the dopaminergic terminals damaged in thestriatum of hemiparkinsonian rats. Since this route of administration ofLGF also promotes a significant behavioural improvement, we propose LGFas a novel factor for use in the treatment of Parkinson's disease.

Example 2 Stimulation of the Serotonin Pathway in the Brain by Injectionof LGF by the Intraperitoneal Route

The experiments conducted for demonstrating the stimulating activity ofLGF, injected by the intraperitoneal route (i.p.) in normal rats, on theserotonin pathway in the brain, are described below.

The possible effect of LGF on the serotoninergic activity in the ratstriatum, hypothalamus and hippocampus was investigated. The animalswere treated with LGF (5 μg/ratXday, intraperitoneal) on the two daysprior to sacrifice. The group of control animals were injected withsaline solution in a volume identical to that of the experimentalanimals.

The serotoninergic activity was evaluated from the accumulation of 5-HTP(5-hydroxytryptophan) after inhibiting decarboxylase with the compoundNSD-1015 (Sigma Co.). This compound was administered (125 mg/kg, i.p.)to the two groups of animals that were sacrificed one hour afteradministration of LGF or saline. The animals were decapitated and thebrain was quickly extracted on ice, followed by dissection of the rightstriatum and right hippocampus. The rest of the brain was kept frozenfor later dissection of the hypothalamus. The tissue samples werehomogenized by sonication in CIO₄H 0.4 N with 0.002% of ascorbic acid,they were centrifuged and the supernatant was used for determining the5-HTP content by reversed-phase high-performance liquid chromatography(HPLC) and electrochemical detection. The methods are described indetail in Marco et al., 1979, Marco et al., 1999. The precipitate wasredissolved in 0.5 N NaOH for protein determination and the finalconcentration of 5-HTP was obtained in relation to the latter. Theresults were expressed as mean value±SEM.

The results, which are shown in Table 1, indicate a significant increasein 5-HTP accumulation both in the striatum and in the hippocampus or inthe hypothalamus after the animals were treated with LGF, which isinterpreted, functionally, as an increase in serotoninergic activity inthese areas of the brain.

TABLE 1 Levels of 5-http in the brain of animals treated with LGF n(number of TISSUE TREATMENT 5-HTP (pmol/mg prot.) samples) StriatumControl 10.88 ± 0.71  12 LGF 13.20 ± 0.75* 12 Hippocampus Control 8.03 ±0.35 12 LGF  10.30 ± 0.60** 12 Hypothalamus Control 26.55 ± 0.99  12 LGF30.13 ± 1.35* 12 *p < 0.05 compared with the control **p > 0.005compared with the control

In view of the activity of LGF on Parkinson's disease, injected by theintraperitoneal route, as well as the activity on the serotonin pathway,which might imply its action in several very different situations, it isto be hoped that LGF has regenerative and reparative activity in otherneurodegenerative diseases, such as Huntington's disease, amyotrophiclateral sclerosis, ataxias etc.

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1. A method of manufacturing a medicinal product suitable forpleiotropic tissue regeneration of at least one damaged tissue, themethod comprising adding liver growth factor (LGF), or lyophilized LGF,to a pharmaceutically acceptable aqueous vehicle, wherein the at leastdamaged tissue forms part of the central nervous system and themedicinal product is designed to be administered systemically.
 2. Themethod according to claim 1, wherein the medicinal product is suitablefor treating a neurodegenerative disease.
 3. The method according toclaim 2, wherein the medicinal product is suitable for of treatingParkinson's disease.
 4. The method according to claim 2, wherein themedicinal product is suitable for treating a neurodegenerative diseasethat is selected from Huntington's disease, amyotrophic lateralsclerosis, or ataxia.
 5. The method according to claim 1, wherein themedicinal product is designed for individuals with an intact blood-brainbarrier.
 6. The method according to claim 1, wherein the medicinalproduct is designed to be administered to a human being.
 7. The methodaccording to claim 1, wherein the medicinal product is designed to beadministered by an intraperitoneal route.
 8. The method according toclaim 1, wherein the medicinal product is designed to be administered byan intravenous route.
 9. A method of treating a disorder or diseasewhose severity or risk of deterioration decreases with the regenerationof at least one tissue, said method comprising administering aneffective amount of liver growth factor to a human being or an animal inneed thereof by a systemic route wherein said disorder or disease isselected from those associated with the central nervous system.
 10. Themethod according to claim 9, wherein the administering is by anintraperitoneal route.
 11. The method according to claim 9, wherein theadministering is by an intravenous route.
 12. The method according toclaim 9, wherein the disease is a neurodegenerative disease.
 13. Themethod according to claim 9, wherein the disease is a Parkinson'sdisease.
 14. The method according to claim 9, wherein the disease isselected from Huntington's disease, amyotrophic lateral sclerosis, orataxia.
 15. The method according to claim 9, wherein the human or animalhas an intact blood-brain barrier.
 16. A method of regeneratingpleiotropic tissue of at least one damaged central nervous systemtissue, the method comprising administering an effective amount of livergrowth factor (LGF) to a subject in need thereof.
 17. The methodaccording to claim 16, wherein the administering is systemic.
 18. Themethod according to claim 16, wherein the administering isintraperitoneal.
 19. The method according to claim 16, wherein theadministering is intravenous.
 20. The method according to claim 16,wherein the subject has an intact blood-brain barrier.